Approximately 60% of all proteins are glycoproteins, which makes glycosylation the most important post-translational modification of individual proteins. Glycans have a high degree of microheterogeneity due to the non-encoded action of enzymes responsible for biosynthesis and the processing of glycan structures bound to proteins.
The knowledge of the glycosylation pattern has an increasing interest not only in the field of glycomics, which deals with the high-throughput analysis of glycans and their functions in complex systems, but also for the biotechnological industry, in which the glycosylation of proteins for therapeutic use has to be controlled for regulatory purposes, for optimizing the function and reuptake or for preventing immunogenic reactions. The unique glycoforms of therapeutic proteins with optimized properties are accessible either by means of chemical approaches using purified or synthetic sugars or by means of fermentation in expression systems.
Methods of analysis of glycoproteins based on mass spectrometry, immunotransfer and chromatography are laborious and unsuitable for the rapid and systematic evaluation of the glycosylation state of proteins.
German Democratic Republic patent DD159569B describes a method for the detection of glycosylated proteins which comprises contacting the sample which supposedly contains the glycoprotein to be detected with an array in which a lectin showing affinity for said protein has been immobilized. However, this method only allows the identification based on a single lectin-glycoprotein interaction and does not allow the simultaneous analysis of several lectins or of several glycoproteins.
Pilobello et al. (ChemBioChem 2005, 6, 985-989), Kuno et al. (Nature Methods, 2005, 2:851-856) and Rosenfeld et al. (J. Biochem. Biophys. Methods, 2007, 70:415-426) have described methods for the characterization of the sugars present in a glycoprotein of interest consisting of using a support in which a series of lectins organized in the form of an array have been immobilized and contacting said array with the glycoprotein of interest labeled fluorescently. The terminal sugar content of the glycoprotein can then be determined by means of detecting (either by fluorescence or by fluorescence-assisted evanescent field) of the point of the array at which fluorescence is observed, indicative of the fact that there has been interaction between the lectin and the glycoprotein. However, this method requires previously isolated glycoproteins therefore it is not suitable for characterizing the glycoprotein content of a complex sample.
US20030153013 describe a method for the simultaneous identification of several proteins with the capacity to bind to respective capture proteins which comprises preparing a support in which said capture proteins organized in the form of an array are immobilized. The sample in which the proteins are located is passed through said support and the presence of proteins in the cells of the array in which the capture proteins are immobilized is detected. Nevertheless, this method does not contemplate the possible use of lectins as capture proteins for studying glycoproteins.
WO02083918 describes arrays of carbohydrates for the identification of proteins with affinity for said carbohydrates (lectins).
Nevertheless, there is a need in the art for methods and reagents for the study of glycoproteins which can be applied in complex samples in which there are different glycoproteins.